FIELD OF THE INVENTION
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This invention relates to cell therapy and in particular to products and methods for use in somatic cells therapy such as stem cell therapy.
LIST OF REFERENCES
The following references are considered to be pertinent for the purpose of understanding the background of the present invention:
1. WO 03/020874, Improved Method for Freezing Viable Cells;
2. U.S. Pat. No. 5,827,741, Cryopreservation of Human Adult and Fetal Pancreatic Cells and Human Platelets;
3. U.S. Pat. No. 6,723,497, Therapeutic Platelets and Methods;
4. Chen et al. 2001, Beneficial effect of intracellular trehalose on the membrane integrity of dried mammalian cells. Cryobiology 43(2):168-81.
5. Crowe et al. 2003, Stabilization of membranes in human platelets freeze-dried with trehalose. Chem. Phys. Lipids. 122(1-2):41-52.
6. Fujiki et al. 1999, Mechanistic Findings of Green Tea as Cancer Preventive for Humans. Proc. Soc. Exp. Biol. Med. 220(4) 225-228;
7. Kumazawa et al. 2004, Direct evidence of interaction of a green tea polyphenol, epigallocatechin gallate, with lipid bilayers by solid-state Nuclear Magnetic Resonance. Biosci Biotechnol Biochem. 68, 1743-7.
8. Suganuma et al. 1999, Green Tea and Chemprevention Mutation Research 428, 339-344.
9. Sherry Chow et al. 2001, Cancer Epidemiology, Biomarkers & Prevention 10, 53-58
10. FDA, CBER, 1998, Guidance for human somatic cell therapy and gene therapy;
11. US 20050020524, Boyd R L. Hematopoietic stem cell gene therapy
12. U.S. Pat. No. 6,887,704. Peled T, et al. Methods of controlling proliferation and differentiation of stem and progenitor cells
13. US 20050118712, Tsai, M S. Two-stage culture protocol for isolating mesenchymal stem cells from amniotic fluid
14. US 20050042754, Miyazaki J. Induction of the formation of insulin-producing cells via gene transfer of pancreatic beta-cell-associated transcriptional factor
15. US 20050008623, Bechetoille N., et al. In vitro production of dendritic cells from CD14+ monocytes
16. US 20050095228, Fraser J K Methods of using regenerative cells in the treatment of peripheral vascular disease and related disorders
17. US Patent application 20050142118, Wernet P. Human cord blood derived unrestricted somatic stem cells (USSC)
18. US 20050059152, Tanavde V. In vitro culture of mesenchymal stem cells (MSC) and a process for the preparation thereof for therapeutic use
19. US 20040197310, Sanberg P R, et al. Compositions and methods for using umbilical cord progenitor cells in the treatment of myocardial infarction
20. Danon D, Madjar J, Edinov E, Knyszynski A, Brill S, Diamantshtein L, Shinar E. Treatment of human ulcers by application of macrophages prepared from a blood unit. Exp Gerontol. 1997 November-December; 32(6):633-41.
21. US 2004191754, Uri M. et. al. Method for Freezing Viable Cells.
22. WO 01/23532, Tsakas S., and Linardos N. Cryopreserved amniotic human cells for future therapeutic, diagnostic, genetic and other uses.
23. YU, J., LIU, J. H., PU, L. Q., CUI, X., WANG, C., OUYANG, S. L., GAO, D. Freeze-drying of Human Red Blood Cells: Influence of Carbohydrates and Their Concentrations. Cell Preservation Technology. 2004; 2(4):270-5.
24. U.S. Pat. No. 6,146,890, Danon David. Method and system for cultivating macrophages.
25. Xaio, H. H., Hua, T. C., Li, J., Gu, X. L., Wang, X., W Meng, L. R., Gao, Q. R., Chen, J., Gong, Z. P. (2004) Freeze-drying of mononuclear cells and whole blood of human cord blood. Cryoletters; 25(2):111-120.
26. Jun Lil, Tse-Chao Hual, Xue-Lian Gul, Yu Dingl, Ming Luol, Hong-Hai Xiaol, Zhi-Jiang Wu, Lv-Rong Meng, Qi-Rong Gao and Jian Chen; Morphology study of freeze-drying mononuclear cells of human cord blood. CiyoLetters 26 (3), 193-200 (2005)
27. Avigdor, A., Goichberg, P., Shivtiel, S., Dar, A., Peled, A., Samira, S., Kollet, O., Hershkovitz, R., Alon, R., Hardan, I., Ben-Hur, H., Naor, D., Nagler, A., Lapidot, T. (2004) CD44 and hyaluronic acid cooperate with SDF-1 in the trafficking of human CD34+ stem/progenitor cells to bone marrow. Blood; 103(8):2981-2982.
28. Ptak, G., Clinton, M., Tischner, M., Barboni, B., Mattioli, M., Loi, P. (2002) Improving delivery and offspring viability of in vitro-produced and cloned sheep embryos. Biology of Reproduction; 67(6):1719-25.
29. WO 0201952
30. Ryan J. M. et al. (2005) Mesenchymal stem cells avoid allogeneic rejection. Journal of Inflammation 2:8;
31. Cord Blood Bank Standard Operating Procedures, Chapter 4, 05/97—Amended 07/99; https://web.emmes.com/study/cord/bld-ch04.pdf
BACKGROUND OF THE INVENTION
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Somatic cellular therapy or cell therapy, including gene therapy, already has great importance in medicine. With technological and scientific advancement the role of cell therapy and its possible applications in medicine are likely to increase.
There are currently numerous examples of work being done in the field of cell therapy. One of the most common applications is bone marrow transplantation. In this kind of cell therapy hematopoietic stem cells (HSC) are taken from a donor bone marrow, umbilical cord blood (UCB) or peripheral blood and transfused into a patient. The HSC migrate into the recipient bone marrow where some of the HSC remain as a continuous population of HSC and others differentiate into new and healthy blood cells. This treatment can be autologous, xenogeneic or allogeneic.
Examples of the types of work being done in research in cell therapy include:
Use of UCB as a source for HSC for treatment of patients whose immune systems have been damaged, such as in US patent application publication No. 2005/0020524,
Ex vivo expansion of somatic cells such as in U.S. Pat. No. 6,887,704 and US patent application publication No. 2005/0142118.
The use of somatic cells as study models for the assessment of immunotoxicity/immunotolerance, for the development of cosmetic and pharmaceutical active principles and for the development and implementation of methods of cell and tissue therapy (e.g. US patent application publication Nos. 2005/0118712, 2005/0042754, 2005/0008623, and 2005/0095228).
Culturing mesenchymal stem cells (MSC) and their preparation for therapeutic use such as in US patent application publication No. 2005/0059152. Amongst the published uses for MSC is the grafting of a donor's MSC to a recipient of an organ (from the same donor, in order to prevent graft rejection (immunosuppresion) (Ryan et al., 2005).
Use in treating circulatory disorders and heart problems such as in US patent application publication No. 2004/0197310.
The use of macrophages in wound healing (Damn et al., 1997).
Preservation at a temperature below 0° C. (defined herein as “cryopreservation”), allows for long storage times and may be at any temperature below 0° C., including such temperatures below −20° C., −70° C., −135° C., or in liquid nitrogen. Cryopreservation is achievable by freezing or by vitrification. In vitrification, ice-crystals are not formed, however high concentrations of cryoprotectant agents that are known to be toxic must be added to the biological material. These cryoprotectant agents must be removed before the biological sample is used, in order not to harm the recipient of the biological material. Freezing is also known to cause damage. For example, ice crystals forming in the solution exert extra-cellular mechanical stress. Intracellular stress can be caused for example by osmosis of water into the extra-cellular space, to replace water that is already frozen.
One factor that has a major effect on the success of cryopreservation is the composition of the solution in which the biological material is immersed prior to freezing. Currently many different cryopreservation solutions are known. Normally, such solutions contain a balanced salt solution such as phosphate buffered saline (PBS), cryoprotectant agents (CPAs), and other molecules including butandiol and methanol.
In addition, sugars, proteins, carbohydrates such as hydroxy ethyl starch (HES), dextran, proteins (especially serum proteins such as albumin) and other macromolecules are also used and are generally termed herein “cryoprotectants”. Trehalose, for example, is thought to be protective by binding to lipid polar groups and replacing water. An example for a currently used storage technique may be found in International patent application publication No. WO 01/23532. Currently used cryoprotectant agents, namely, DMSO, ethylene glycol, glycerol and other polyalcohols, are toxic to cells and therefore upon thawing need to be removed or even washed. Use of serum may also be hazardous, since there is a hazard of contamination (especially when the source is human) or if the source is non-human (e.g. bovine) there are also health hazard (e.g. prions and ill match of the cells to the human body).
In most cryopreservation protocols, preservation of the frozen biological material is at a temperature below −130° C. This is normally done in containers of liquid nitrogen (LN) by either immersion of the biological material in LN or in LN vapor. This adds significantly to the cost of long-term preservation. In addition, incidents are known where the LN in the container evaporated (either due to a malfunction of the container or human error) and the biological materials were damaged. Furthermore, when storing in LN cross contamination can occur. This might be discovered only after use and cost also in patients\' lives.
Moreover, in many applications, the cell therapy technique includes a step of processing the cells and usually such processed or treated cells are more sensitive to preservation in cold temperatures. The result is that the cells have a very short shelf life after the process is completed and the cells must be administered to a patient (e.g. injected) or otherwise used in a very short time, sometimes even a few hours after the process is completed. This limitation causes the market of providing treated or processed cells to be considered as a “service”. A good long term preservation method will allow the treated cells to be regarded more as a “product”, ready to be used whenever required and not only when their processing is completed.
One method that can overcome these obstacles is lyophilization of the frozen biological material (e.g. as described in US patent application publication No. 2004-191754). Lyophilization is a process in which ice crystals are removed by sublimation and desorption, resulting in dry, or partially dry, matter. The lyophilized material may be stored at room temperature for a long period of time and be rehydrated for use by simply adding water. Lyophilization results in higher survival rates than air drying or heating, but is still a damaging process.
In order to enhance the biological material\'s ability to survive the freeze-drying process, intercellular and/or extra-cellular lyoprotectant agents (LPAs) are often added to the biological material. Carbohydrates and polymers (such as PVP, Dextran, Hydroxy Ethyl Starch (HES), glucose, sucrose, mannose, lactose, trehalose and other) are known to be used for stabilization of the cells during lyophilization and storage in the dry state (Yu et al., 2004).
One method of lyophilization of cells includes introduction of trehalose into the cells. Trehalose is known to protect cell membranes in a dry state (Chen et al., 2001). It was also shown to improve platelet survival after freeze-drying (Crowe et al., 2003).
Umbilical cord blood (UCB) is a source for hematopoietic stem cells (HSC). HSC are cells that can differentiate into all blood cells. Other sources for HSC are bone marrow and a very small amount of HSC can be found circulating in peripheral blood (as WBC). Morphologically, HSC have a round nucleus similar to the mononuclear white blood cells (lymphocytes and monocytes). They resemble lymphocytes very much, and may be slightly bigger. The method to differentiate between them is according to cell membrane antigens. HSC are normally identified by expression of the CD34 antigen. HSC (from peripheral blood, bone marrow or UCB) are given to patients whose immune systems have been damaged, e.g. due to chemotherapy and/or radiotherapy and in different diseases such as: acute and chronic leukemias, myelodysplastic syndromes, Hodgkin lymphoma, non-Hodgkin lymphoma, and multiple myeloma, aplastic anemia, thalassemia, sickle cell anemia, neuroblastoma and more.
The current method for the preservation of somatic cells is using 5-10% DMSO and storage in liquid nitrogen (LN). When storing SC from UCB and from peripheral blood the cells are separated using ficoll-paque and the fraction that is stored are the MNC (Cord Blood Bank Standard Operating Procedures, Chapter 4).
Lyophilization of mononuclear cells derived from human umbilical cord blood was described by Xiao, H. H., et al., 2004 and Lil et al. 2005.
Epigallocatechin Gallate (EGCG)
Epigallocatechin gallate (EGCG) is a polyphenol (MW 458.4) found naturally for example in green and black tea. The well-known beneficial effects associated with such tea are attributed, at least in part, to EGCG. Among the mechanisms associated with EGCG\'s beneficial effects are its ability to function as an antioxidant, its ability to associate with the phospholipids bi-layer of the cell membrane (Fujiki et al. 1999) and the lipid head groups of liposomes (Kumazawa et al., 2004) and more. Whilst EGCG is the main constituent of green tea, other polyphenols that are found naturally in green tea, such as epicatechin gallate (ECG) epigallocatechin (EGC) and epicatechin (EC), are also found in green tea and, like EGCG, are considered to be non-toxic. These polyphenols share structural and functional properties with EGCG (Suganuma et al. 1999).
International patent application Publication No WO02/01952 describes a preservation fluid for cells and tissues, containing a polyphenol as the active ingredient. The fluid may further contain trehalose.
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OF THE INVENTION
The present invention is based on the inventors\' surprising discovery that preservation by either freezing or freeze drying of somatic cells, which have either undergone manipulation or not (as described below), and subsequent reconstitution of said cells resulted in viable, functioning post-preservation cells that can be used for the purposes of cell therapy.
The present invention is further based on the finding that preserved mononuclear cells composed mainly of stem cells can be reconstituted and transplanted into an animal, while maintaining their viability and functionality as indicated by their ability to increase the survival rate of immune-compromised animals, and their ability to produce circulating blood cells carrying the graft cell\'s genetic material.
Thus, the present invention provides, by a first of its aspects, a preserved cell preparation, essentially free of one or more members of a group of cryoprotecing agents consisting of polyalcohols, DMSO and cryoprotecting proteins, the preserved cell preparation comprising somatic cells and at least one polyphenol, wherein upon reconstitution of cells in the cell preparation, at least a portion of said stem cells are viable, said portion being sufficient for use of the cell preparation in somatic cell therapy. Preferably, the cell preparation is free of polyphenol and DMSO and does not require the addition of serum proteins (e.g. albumin, known as a cryoprotecting protein).
In accordance with a second aspect, the present invention provides the use of a preserved cell preparation for the production of a therapeutic composition for somatic cell therapy, the preserved cell preparation being essentially free one or more members of a group of cryoprotecing agents consisting of polyalcohols, DMSO and cryoprotecting proteins, the preserved cell preparation comprising somatic cells, wherein upon reconstitution of cells in the cell preparation, at least a portion of said somatic cells are viable, said portion being sufficient for use of the therapeutic composition in somatic cell therapy.
In accordance with a third aspect, the present invention provides a reconstituted cell preparation being essentially free of one or more members of a group of cryoprotecing agents consisting of polyalcohols, DMSO and cryoprotecting proteins, the preserved cell preparation comprising post-preservation somatic cells and at least one polyphenol, wherein at least a portion of said post-preservation somatic cells are viable, said portion being sufficient for use of the reconstituted cell preparation in somatic cell therapy. According to a preferred embodiment, the reconstituted cell preparation is essentially free of polyalcohol, DMSO and serum proteins (e.g. albumin).
In accordance with a fourth aspect, the present invention provides a method of treating a subject in need of somatic cell transplantation, the method comprises administering to said subject a cell preparation essentially free of one or more members of a group of cryoprotecing agents consisting of polyalcohols, DMSO and cryoprotecting proteins, the preserved cell preparation comprising a effective amount of post-preservation and viable somatic cells the amount of the viable somatic cells being sufficient for re-establishing the stem cells in the subject\'s body.
In accordance with one embodiment, the somatic cells are stem cells, preferably human stem cells (hSC).
BRIEF DESCRIPTION OF THE DRAWINGS
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In order to understand the invention and to see how it may be carried out in practice, preferred embodiments will now be described, by way of non-limiting example only, with reference to the accompanying drawings, in which:
FIGS. 1A-1B are bar graphs showing membrane integrity (FIG. 1A) and percentage (FIG. 1B) of cells after freeze thawing or freeze drying with different solutions: HSA & Trehalose (1) HSA, Trehalose & EGCG (2) HSA & EGCG (3) Trehalose & EGCG (4) Dextran & EGCG (5) (results in FIG. 1A are shown as the percentages of treated samples compared to the fresh samples).
FIGS. 2A-2B are bar graphs demonstrating the percent of membrane integrity of samples after freeze thawing and freeze drying as compared to the fresh samples (set as a 100%) using a freezing solution comprising 0.4M trehalose (FIG. 2A) or 0.1M trehalose (FIG. 2B). The graphs show an EGCG dose response on UCB derived MNC.
FIG. 3 is a graph showing the proliferation of mononuclear cells (MNC) after freezing and thawing and after lyophilization and re-hydration, according to some embodiments of the invention. (×2.5 EGCG=1.03 mM; ×10 EGCG=4.12 mM)
FIGS. 4A-4B are graphs showing membrane integrity (FIG. 4A) and percentage of cells that are metabolizing (FIG. 4B) after freezing at different cooling rates prior to thawing or drying (results are shown as the percentages of treated samples compared to the fresh samples).
FIG. 5 is a photograph of CFU-GM colonies grown from previously lyophilized MNC as viewed by a light microscope.
FIGS. 6A-6C are bar graphs showing the percentage of membrane integrity in macrophages that were frozen and thawed and macrophages that were freeze dried with different trehalose concentrations where macrophages were separated on Ficoll-Paque gradient prior to freezing (FIG. 6A) or without prior separation on Ficoll-Paque gradient (FIG. 6B); or tells were frozen as whole macrophage units (FIG. 6C).
FIG. 7 is a bar graph demonstrating the survival patterns of mice injected with bone marrow derived MNC compared with mice that were not administered with NMC.
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OF THE INVENTION
Some terms used herein and their meanings are as follows:
Somatic cells—any sample that comprises cells of an organism, which are not gametes. Such cells include stem cells, white blood cells (WBC), umbilical cord blood (UCB) cells (which are not stem cells) or fractions thereof, embryos, embryonic stem cells, bone marrow cells and other mononuclear cells (MNC), such as lymphocytes or a combination of any of the above. Such cells may be taken from a mammal, e.g. humans.
Stem cells—cells that have the capacity to replicate themselves into cells with similar properties in order to maintain a pool of precursor cells which may then differentiate to produce specialized cell types. Such stem cells may comprise, without limitation, any of the following: pluripotent stem cells, totipotent stem cells, and unipotent stem cells, adult stem cells, embryonic stem cells, hematopoietic stem cells (FISC), mesenchymal stem cells (MSC) and stromal stem cells. HSC may give rise to cells of the lymphoid lineage and to cells of the myeloid lineage. HSC may be obtained from any source, including isolation from mononuclear cells found in Umbilical cord blood (UCB), bone marrow or the peripheral blood. Such cells may be taken from a mammal, e.g. humans and human derived stems cells.
Cellular therapy or cell therapy—administration to humans of autologous, allogeneic, or xenogeneic living cells for any purpose including diagnostic or preventive purposes (Guidance for Industry-Guidance for Human Somatic Cell Therapy and Gene Therapy, FDA, Center for Biologics Evaluation and Research (CBER), March 1998), and healing or treatment of any condition or malady, for example regenerative medicine.
For the purpose of this invention, the term cell therapy also includes the process of therapeutic cloning which means the use of stem cells to produce cloned embryo that serves as a source for embryonic stem cells used for cell therapy. In the framework of cellular therapy use may be made of any appropriate somatic cells as known in the art, including but not necessarily manipulated cells. Cellular therapy may also include the use of stem cells for production of tissue or organ for transplantation including the use of the cells for therapeutic cloning (e.g. as a source for nuclei in nuclear transfer).